Mycobacterium tuberculosis is the leading cause of bacterial infectious disease deaths worldwide. Drug-resistant strains (MDR and XDR-TB) are an emerging problem and could lead to significant issues in the US because over half of the cases are in foreign born persons. Biotin biosynthesis has recently been identified as an attractive target for the development of new ant tubercular agents. In this proposal we describe the design, development, and optimization of a novel HTS-assay for BioA, which catalyzes the second step of biotin biosynthesis in Mtb. The objectives of this application are to screen BioA using our novel HTS assay and then perform secondary and tertiary screening of identified hits. In the first specific aim, we will prepare chemical and biochemical reagents for the primary and secondary assays. In the second specific aim, we will develop and screen hits using an orthogonal LC-MS based assay. In the third specific aim, we will perform tertiary screening using mutant strains of M. smegmatis that over- and under-express BioA to access biological activity and characterize on-target specificity.